BIOSYNTHESIS OF MYCOBACTERIAL IMYCOCEROSATE ESTER VIRULENCE FACTORS. Obligate mycobacterial pathogens and opportunistic mycobacterial pathogens within the Mycobacterium tuberculosis complex or outside of this complex (i.e., non-tuberculosis mycobacteria [NTM]) are responsible for substantial morbidity and mortality worldwide. Treatment of mycobacterial infections is complicated by the resilience of mycobacteria to many antibiotics afforded by the permeability barrier of the unique mycobacterial cell wall. Mycobacterial infections are becoming increasingly difficult to treat due to the rise of acquired drug resistance as well. Multidrug-resistant and extensively drug-resistant strains of M. tuberculosis pose a global menace. Multidrug-resistant strains of M. leprae threaten to compromise the future of leprosy control. Infections of NTM are on the rise, and drug resistant NTM are a growing concern in the USA and abroad. Development of the rich therapeutic arsenal needed to counter the rise of drug resistant mycobacteria requires exploitation of conventional and unconventional drug targets and therapeutic approaches. In this light, the mycobacterial enzymes needed for the biosynthesis of (glyco)lipids required for virulence and involved in the fortification of the cell wall permeabilit barrier are target candidates for exploring the development of innovative adjuvant drugs to be added to multidrug treatments to improve clinical outcomes. Mycobacterial dimycocerosate esters (DIMs) are a group of free lipids and glycolipids (hereinafter referred to as PDIMs and PGLs, respectively) unique to the outer membrane of many pathogenic mycobacteria (e.g., M. tuberculosis complex, M. leprae and several NTM). DIMs are major virulence factors that down-regulate and subvert immune mechanisms, strengthen the cell wall permeability barrier, reduce drug susceptibility and, possibly, afford a layer of protection against the oxidative defense in th macrophage of the host. The proposed project will utilize genetic and biochemical approaches to deliver new mechanistic knowledge on the biosynthesis of DIMs. The project will be pursued via a focused aim with four sub aims and using the opportunistic pathogen M. marinum as a representative of DIM producers. M. marinum, the closest genetic relative of the M. tuberculosis complex, is often utilized to model aspects of M. tuberculosis complex pathogenesis and offers greater experimental tractability than other DIM producers. Dissecting DIM biosynthesis will pave the way to the long-term goal of exploring the development of innovative adjuvant drugs that block DIM synthesis, thus having the potential to empower host defenses and (hyper)sensitizing DIM-producing mycobacteria to some antimicrobial drug treatments.